Conventional methods of isolation of mRNA isolate total mRNA from the cell. The mRNA isolated this way includes cytosolic mRNA and nuclear mRNA. For the purposes of quantitation and analysis of expression, it is important to only isolate the newly synthesized mRNA, not that which is being degraded in the cytoplasm. In this way one gets a “true” reading of the expression level or the changes in expression of a gene.
Gene expression analysis is of major interest in the field of functional genomics. Now that the human genome has been sequenced, there is a great need to identify genes and mutations which are involved in disease. These may be genes which are up- or down-regulated or genes which have polymorphisms. Many of the methods used to analyze these genes involve the isolation of mRNA. For example, DNA microarray chips can analyze the expression profile of thousands of different genes simultaneously. Reverse transcription polymerase chain reaction (RT-PCR) is a very sensitive method used to quantitate the expression of individual genes, and is rapidly replacing the more labor-intensive Northern blot analysis. There are many other non-PCR technologies available for sensitive gene detection, such as Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), and branched DNA (bDNA) amplification.
Although many of these and other detection technologies have been developed and commercialized, the starting material is always total RNA or poly(A)+ RNA from cells or tissue samples. When total RNA or mRNA from a whole cell is used, the results are a mixture of the products of transcription as well as degradation products from the cytosol. This makes it difficult to detect expression of new mRNA under conditions that digestion of mRNA is in progress. Further, when a large amount of mRNA is already present, it is difficult to detect a slight change in the quantity of mRNA, and thus sensitivity is low.
The transcription of immature mRNA occurs in the nucleus, where poly(A) tails are attached and splicing occurs as needed. After this, the mature mRNA molecules then migrate into the cytoplasm and are translated and degraded. Since the amount of mRNA in the nucleus is directly related to the amount of transcription—not degradation—of mRNA, attempts have been made to purify the nuclear fraction in order to measure the amounts of specific mRNA (or nuclear RNA) in the nuclear fraction. Previous attempts to specifically analyze the nuclear fraction include the nuclear run-on assay which measures the level of transcription of specific genes. However, a major obstacle to the use of this assay is the amount of time required, the need for very careful handling to avoid ribonuclease contamination, and the generation of radioactive waste. Other techniques for purification of the nuclear fraction used time-consuming ultracentrifugation or labor-intensive microinjection to perform nuclear mRNA purification and analysis. These methods are not high throughput, and not suitable for precise quantitation. Moreover, it is not clear whether nuclear mRNA is preserved, for example, during the ultracentrifugation.
Thus, a rapid, sensitive, reliable, and high throughput methodology for isolation and analysis of the nuclear fraction of mRNA is needed. This would allow the analysis of “true” transcription levels of a gene.